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1.
Stem Cells Int ; 2024: 3601101, 2024.
Article in English | MEDLINE | ID: mdl-38737365

ABSTRACT

Long-term diabetes often leads to chronic wounds refractory to treatment. Cell-based therapies are actively investigated to enhance cutaneous healing. Various cell types are available to produce biological dressings, such as adipose-derived stem/stromal cells (ASCs), an attractive cell source considering their abundancy, accessibility, and therapeutic secretome. In this study, we produced human ASC-based dressings under a serum-free culture system using the self-assembly approach of tissue engineering. The dressings were applied every 4 days to full-thickness 8-mm splinted skin wounds created on the back of polygenic diabetic NONcNZO10/LtJ mice and streptozotocin-induced diabetic K14-H2B-GFP mice. Global wound closure kinetics evaluated macroscopically showed accelerated wound closure in both murine models, especially for NONcNZO10/LtJ; the treated group reaching 98.7% ± 2.3% global closure compared to 76.4% ± 11.8% for the untreated group on day 20 (p=0.0002). Histological analyses revealed that treated wounds exhibited healed skin of better quality with a well-differentiated epidermis and a more organized, homogeneous, and 1.6-fold thicker granulation tissue. Neovascularization, assessed by CD31 labeling, was 2.5-fold higher for the NONcNZO10/LtJ treated wounds. We thus describe the beneficial impact on wound healing of biologically active ASC-based dressings produced under an entirely serum-free production system facilitating clinical translation.

3.
Int J Mol Sci ; 24(16)2023 Aug 08.
Article in English | MEDLINE | ID: mdl-37628718

ABSTRACT

Tissue-engineered skin substitutes (TESs) are used as a treatment for severe burn injuries. Their production requires culturing both keratinocytes and fibroblasts. The methods to grow these cells have evolved over the years, but bovine serum is still commonly used in the culture medium. Because of the drawbacks associated with the use of serum, it would be advantageous to use serum-free media for the production of TESs. In a previous study, we developed a serum-free medium (Surge SFM) for the culture of keratinocytes. Herein, we tested the use of this medium, together with a commercially available serum-free medium for fibroblasts (Prime XV), to produce serum-free TESs. Our results show that serum-free TESs are macroscopically and histologically similar to skin substitutes produced with conventional serum-containing media. TESs produced with either culture media expressed keratin 14, Ki-67, transglutaminase 1, filaggrin, type I and IV collagen, and fibronectin comparably. Mechanical properties, such as contraction and tensile strength, were comparable between TESs cultured with and without serum. Serum-free TESs were also successfully grafted onto athymic mice for a six-month period. In conclusion, Surge SFM and Prime XV serum-free media could be used to produce high quality clinical-grade skin substitutes.


Subject(s)
Skin, Artificial , Animals , Mice , Culture Media, Serum-Free , Tissue Engineering , Fibroblasts , Keratinocytes , Mice, Nude
4.
Int J Mol Sci ; 24(3)2023 Jan 17.
Article in English | MEDLINE | ID: mdl-36768144

ABSTRACT

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.


Subject(s)
Keratinocytes , Tissue Engineering , Animals , Mice , Humans , Keratinocytes/metabolism , Skin/metabolism , Epidermis/metabolism , Epidermal Cells , Culture Media, Serum-Free/pharmacology , Cells, Cultured
5.
Biomed Mater ; 17(5)2022 08 11.
Article in English | MEDLINE | ID: mdl-35950736

ABSTRACT

The increasing need for tissue substitutes in reconstructive surgery spurs the development of engineering methods suited for clinical applications. Cell culture and tissue production traditionally require the use of fetal bovine serum (FBS) which is associated with various complications especially from a translational perspective. Using the self-assembly approach of tissue engineering, we hypothesized that all important parameters of tissue reconstruction can be maintained in a production system devoid of FBS from cell extraction to tissue reconstruction. We studied two commercially available serum-free medium (SFM) and xenogen-free serum-free medium (XSFM) for their impact on tissue reconstruction using human adipose-derived stem/stromal cells (ASCs) in comparison to serum-containing medium. Both media allowed higher ASC proliferation rates in primary cultures over five passages compared with 10% FBS supplemented medium while maintaining high expression of mesenchymal cell markers. For both media, we evaluated extracellular matrix production and deposition necessary to engineer manipulatable tissues using the self-assembly approach. Tissues produced in SFM exhibited a significantly increased thickness (up to 6.8-fold) compared with XSFM and FBS-containing medium. A detailed characterization of tissues produced under SFM conditions showed a substantial 50% reduction of production time without compromising key tissue features such as thickness, mechanical resistance and pro-angiogenic secretory capacities (plasminogen activator inhibitor 1, hepatocyte growth factor, vascular endothelial growth factor, angiopoietin-1) when compared to tissues produced in the control FBS-containing medium. Furthermore, we compared ASCs to the frequently used human dermal fibroblasts (DFs) in the SFM culture system. ASC-derived tissues displayed a 2.4-fold increased thickness compared to their DFs counterparts. In summary, we developed all-natural human substitutes using a production system compatible with clinical requirements. Under culture conditions devoid of bovine serum, the resulting engineered tissues displayed similar and even superior structural and functional properties over the classic FBS-containing culture conditions with a considerable 50% shortening of production time.


Subject(s)
Cell Culture Techniques , Vascular Endothelial Growth Factor A , Adipose Tissue , Cell Culture Techniques/methods , Cell Differentiation , Cell Proliferation , Cells, Cultured , Connective Tissue , Humans , Vascular Endothelial Growth Factor A/metabolism
6.
J Mater Chem B ; 4(20): 3608-3619, 2016 May 28.
Article in English | MEDLINE | ID: mdl-32263390

ABSTRACT

Inflammation is a normal phase of the wound healing process, which likely occurs following tissue transplantation. For reconstructive surgery purposes, engineered adipose tissues represent promising alternatives to autologous fat grafts. It is therefore important to study the impact of an inflammatory microenvironment on the cellular functions of the different cell types comprised within matrix-rich reconstructed tissues. In this study, human reconstructed adipose tissues (hrATs) featuring a preformed capillary network formed by microvascular endothelial cells (hMVECs) were produced from adipose-derived stem/stromal cells (ASCs) by the self-assembly approach of tissue engineering. We hypothesized that a prolonged inflammatory context, mediated by tumor necrosis factor (TNF) and interleukin-1ß (IL-1ß), would impact hrATs' secretory profile and mediate detrimental effects on the microvascular network in vitro. Analysis of conditioned media established tissue responsiveness through the increased secretion of monocyte chemoattractant protein-1 (up to 23 fold), interleukin-6 (up to 69 fold) and angiopoietin-1 (up to 2.7 fold) after 3 and 6 days of cytokine exposure, along with a significant reduction in adiponectin secretion. Imaging of the preformed capillary network within the hrATs revealed increased disorganization in the presence of TNF/IL-1ß, featuring a less extended and less ramified network with apoptotic hMVECs in the remaining capillary structures. These results indicate that a prolonged inflammatory context can be deleterious to the capillary network featured by in vitro engineered tissues. Strategies aiming at preserving the integrity of the vascular network will help develop substitutes that are better suited to face inflammatory conditions upon grafting.

7.
PLoS One ; 10(9): e0137612, 2015.
Article in English | MEDLINE | ID: mdl-26367137

ABSTRACT

Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells, extracellular matrix and differentiated adipocytes, in addition to compounds modulating adipogenesis from precursor cells.


Subject(s)
Adipokines/metabolism , Adipose Tissue/metabolism , Tissue Engineering , Tumor Necrosis Factor-alpha/pharmacology , Adipose Tissue/drug effects , Chemokine CCL2/metabolism , Enzyme-Linked Immunosorbent Assay , Fatty Acid Synthase, Type I/genetics , Fatty Acid Synthase, Type I/metabolism , Gene Expression Regulation , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , Hepatocyte Growth Factor/metabolism , Humans , NF-kappa B/metabolism , Nerve Growth Factor/metabolism , RNA, Messenger/metabolism , Vascular Endothelial Growth Factor A/metabolism
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